Biodegradation of poly(tetramethylene succinate-co-tetramethylene adipate) and poly(tetramethylene succinate) through water-soluble products

Article abstract of DOI:10.1002/etc.5620200501

Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hydrolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were identified by high-performance liquid chromatography-mass spectrometry and ¹H-nuclear magnetic resonance, which indicated that a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover, 20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80 mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native soil for 60 d reached > 60%.

Full text of DOI:10.1002/etc.5620200501

Environmental Toxicology and Chemistry, Vol. 20, No. 5, pp. 941–946, 2001
2001 SETAC
Printed in the USA
0730-7268/01 $9.00 .00
᭧
ϩ
Environmental Chemistry
BIODEGRADATION OF POLY(TETRAMETHYLENE SUCCINATE-CO-
TETRAMETHYLENE ADIPATE) AND POLY(TETRAMETHYLENE SUCCINATE)
THROUGH WATER-SOLUBLE PRODUCTS
E
IICHI
K
ITAKUNI,*† KATSUYUKI
Y
OSHIKAWA,† KATSUHARU
OKOTA,‡ RYOJI
N
AKANO,† JUNJI
S
ASUGA,† MASAYOSHI
AKABE
NOBIKI,†
H
IROSHI
N
AOI,† YOSHIHISA
Y
I
SHIOKA,‡ and YOSHIYUKI
Y
§
†Central Research Lab, Showa Denko K.K., Ohnodai 1-1-1, Midori-ku, Chiba 267-0056, Japan
‡Showa High Polymer Co. Ltd., Kanda-nishiki cho 3-20, Chiyoda-ku, Tokyo 101-0054, Japan
§Kurume Laboratory, Chemicals Evaluation and Research Institute, Chuo-machi 19-14, Kurume, Fukuoka 830-0023, Japan
(
Received April 2000; Accepted 15 November 2000)
7
Abstract—Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hy-
drolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were
identified by high-performance liquid chromatography–mass spectrometry and 1H-nuclear magnetic resonance, which indicated that
a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover,
20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane
compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers
and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis
of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80
mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence
of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of
microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native
soil for 60 d reached
Ͼ 60%.
Keywords—Poly(tetramethylene succinate-co-tetramethylene adipate)
Poly(tetramethylene succinate)
Enzymatic hydrolysis
Oligomers
Biodegradability
INTRODUCTION
MATERIALS AND METHODS
Chemicals
The environmental impact of non-biodegradable waste
polymer materials has become steadily more acute. These ma-
terials find their was into the municipal solid waste stream. In
contrast, biodegradable polyesters are expected to reduce the
amount of waste. However, water-soluble intermediates of
these polyesters are likely to be formed during degradation,
and these intermediates might have environmental impacts.
Evaluating the desirability of using biodegradable polyesters
necessitates identifying and quantifying the intermediates
formed during biodegradation, and determining the half-lives
of these intermediates. We previously showed that some water-
soluble oligomers could be readily obtained from
poly(tetramethylene succinate-co-tetramethylene adipate)
(PBSA) by enzymatic hydrolysis [1]. These oligomers were
considered to be the intermediate products liberated from the
polymer by extracellular enzymes. This experimental tech-
nique seems applicable to other polyesters as well.
In this study, we scaled up the system to provide enough
sensitivity to survey all the hydrolysates from degradation of
PBSA and poly(tetramethylene succinate) (PBS), and evalu-
ated the half-lives of the obtained oligomers. We also focused
on two typical oligomers, and showed that the conversion rates
of the constituent carbons in the polymer to carbon dioxide,
biomass, and dissolved metabolites depend on chemical struc-
ture and the environmental conditions of the microorganisms.
The PBSA and PBS used in this study were supplied by
Showa Highpolymer (Tokyo, Japan). They are commercially
available biodegradable plastics (PBSA, Bionolle 3020; PBS,
Bionolle 1020), consisting of 1,4-butanediol (B), succinic acid
(S), and adipic acid (A) with a composition ratio of 50:40:10
as B:S:A for PBSA and 50:50 as B:S for PBS. The PBSA and
PBS were increased in molecular weight by chain-expansion
with the addition of a slight amount of hexamethylene diiso-
cyanate (H) as coupling agent. The resultant weight-averaged
molecular weight (mol wt) was approximately 100,000 for
both. They were powdered by freezer mill and sifted to 125
␮
m to 250 ␮m before the hydrolysis experiment.
Bis(hydroxybutyl) succinate (BSB) was prepared through
chemical synthesis based on a polycondensation reaction of B
with S at a composition ratio of 50:50 as B:S, and was extracted
with acetone. The extract contained BSB at 50 mol%, B at 24
mol%, BSBSB at 22 mol%, and BSBSBSB at 4 mol%.
Bis(hydroxybutyl) hexamethylene dicarbamate (BHB) was
also chemically synthesized by reacting H and excess amount
of 4-hydroxybutyl methacrylate in anhydrous CH3COOCH3,
followed by reduction of the methacrylic acid with NaOH in
MeOH. The resulting BHB was extracted with acetone and
had a purity of 98 mol%.
Hydrolysis of PBSA or PBS by extracellular lipase
As hydrolyzing enzyme, Lipase Type XII (Chromobacte-
rium viscosum, Sigma, St. Louis, MO, USA) was used with
* To whom correspondence may be addressed
(kitakuni@ctl.sdk.co.jp).
941

942
Environ. Toxicol. Chem. 20, 2001
E. Kitakuni et al.
further purification by dialysis against deionized water. The
PBSA (7.5 g) powder was hydrolyzed in 750 ml of deionized
water by 50 U/ml of the enzyme, maintaining the optimal pH
of 7.5 by the addition of NaOH and maintaining the temper-
932 (LECO, St. Joseph, MI, USA). These techniques assisted
the HPLC-MS analysis in determining the chemical structure.
Biodegradation
ature optimum of 35
der was similarly hydrolyzed in 800 ml of deionized water.
After 20 h of hydrolysis, the residual unreacted polymers were
Њ
C for the enzyme. The PBS (8.0 g) pow-
The biodegradation test was conducted according to the
Ministry of International Trade and Industry (Toyko, Japan)
test [2]. The test substance (30 mg) and 9 mg as mixed liquor
suspended solid of activated sludge were added to 300 ml of
excluded by membrane filter with 0.2-␮m pore size (Millipore
Omnipore Membrane, Millipore, Bedford, MA, USA). The
enzyme was subsequently excluded by ultrafiltration (Minitan,
Millipore). The solution was finally freeze-dried and the ob-
tained water-soluble oligomers were analyzed by high-perfor-
mance liquid chromatography–mass spectrometry (HPLC-
MS).
mineral medium and stirred at 25ЊC for 28 d, during which
time the biochemical oxygen demand (BOD) was measured
continuously using a BOD meter (Coulometer, Ohkura Elec-
tric, Tokyo, Japan). The specific BOD demand of the test ma-
terial was calculated as the difference between oxygen con-
sumption in the test flasks and the blanks divided by the con-
centration of the test material at time t. The percentage bio-
degradation was calculated as the ratio of the specific BOD
demand to the theoretical oxygen demand. Aniline was used
as a positive control.
HPLC-MS and size exclusion chromatography-mass
spectrometry (SEC-MS)
For the evaluation of water-soluble products in the hydro-
lysis experiments of PBSA and PBS, HPLC-MS measurement
was carried out according to Ando et al. [1] with the mass
spectrometer (JMS-SX102A, JEOL, Tokyo, Japan) connected
to a high-performance liquid chromatograph equipped with
frit-fast atom bombardment interface. Separation was per-
Soil burial test
The BHB (10 mg) was well mixed with 13 g of native soil
containing 3 g of mineral water, and placed in a BOD reactor
at 25ЊC for 50 d. The soils used for the biodegradation were
a mixture of volcanic ash soil (Andosol) and red-yellow soil
obtained from the surface of a cultivated field in Ibaraki Pre-
formed at 40ЊC using a Shodex OHpak F-511A (Showa Denko
K.K., Tokyo, Japan) column. Gradient elution was performed
at a flow rate of 1.0 ml/min using two mobile phases containing
0.1% of trifluoroacetic acid (trifluoroacetic acid:acetonitrile
and trifluoroacetic acid:water:acetonitrile). In the first step, the
mobile phase consisted of 5% acetonitrile for 5 min, followed
by a linear gradient which ran from 5 to 80% acetonitrile at
50 min. A 1.0% glycerol solution was added at 0.4 ml/min
after the column as a matrix for fast atom bombardment ion-
ization. For each component, the mass spectrum was measured
and the chemical structure was determined by the pseudomo-
fecture (Japan). The soils were sieved (Ͻ2 mm) and stored at
4
Њ
C before use. The biodegradability was calculated as men-
tioned above.
Determination of a carbon balance
The determination of a total carbon balance was carried out
at the end of biodegradation. The total carbon balance was
based on the summation of the amount of carbon derived from
the following measurements: the carbon evolved as carbon
dioxide, the carbon produced as new biomass, the carbon trans-
formed into water-soluble organic metabolites, the carbon de-
termined as dissolved organic carbon (DOC), and the carbon
remaining in the undegraded polymer material. The carbon
sum was compared with the amount of organic carbon in the
test material introduced into the test system. The BOD evolved
was used to determine the amount of carbon evolved as carbon
dioxide. The culture medium was filtered using silver mem-
ϩ
lecular ion peak (MH ). The amount of each product was
quantified by the peak area of the pseudomolecular ion peak.
The amount of each hydrolysate was quantified as disuccinic
acid ester of 1,4-buthandiol (SBS) equivalent, and the mount
of each monomer was quantified using itself as the standard.
For the evaluation of BSB and BHB in the biodegradation
experiments, SEC-MS measurement was used. The SEC was
performed at 40ЊC using a Shodex OHpak SB-8025HQ (Showa
brane filters of 0.45-
lipore Silver Membrane AG4502500). The filter cake was dried
in vacuum at 105 C for 3 h before weighing. The amount of
carbon in the cake was determined by CHNS analysis using
a LECO CHNS-932 (LECO). The amount of carbon in the
residual materials was determined by SEC-MS. The filtrate
was examined to determine the amount of DOC and dissolved
inorganic carbon using a Shimazu TOC-5000A (Shimazu, To-
kyo, Japan).
␮m pore size and 25-mm diameter (Mil-
Denko K.K.) column and the mobile phase consisted of water:
acetonitrile:acetic acid at a ratio of 60:40:0.05. The eluted
materials were detected by a mass spectrometer (JMS-
SX102A, JEOL, Tokyo, Japan) connected to the size exclusion
chromatograph. The amount of residual BSB or BHB was
Њ
ϩ
determined by the pseudomolecular ion peak (MH ), using
itself as the quantitative standard.
1H-Nuclear magnetic resonance
1
The H-nuclear magnetic resonance was performed using
RESULTS AND DISCUSSION
a Bruker AMX-400 (Billerica, MA, USA) by dissolving the
Ϫ
HPLC-MS analysis of water-soluble products
acquired oligomers into dimethylsulfoxide d6. The resonance
spectrum was completely assigned to the chemical structure
without unknown impurities below 1.0% (wt/wt).
The enzymatic hydrolysis of PBSA produces a mixture of
monomers and oligomers as water-soluble products. The qual-
itative analysis of each product was made mainly by using its
pseudomolecular ion peak. Some of the same molecular weight
oligomers consisting of different monomer units were distin-
guished by both their mass spectra and values of retention
time, whereas some products could not be distinguished, for
example ABSB and SBAB, which have the same monomer
composition and molecular weight. These oligomers are rep-
Fourier transform infrared spectroscopy and chemical
element analysis
Fourier transform infrared spectroscopy was performed
with Perkin Elmer 1650 FTIR (Perkin-Elmer, Norwalk, CT,
USA). Chemical element analysis was performed with a Yan-
agimoto MT-3 (Yanagimoto, Kyoto, Japan) and LECO CHNS-

Biodegradation of PBS/PBSA through water-soluble products
Environ. Toxicol. Chem. 20, 2001
943
tained the small oligomers that were SB, BSB, SBS, BHB,
and SBSB; identical to those from PBSA.
Specificity in biodegradation
Figure 2 shows the percentage biodegradation of BSB and
BHB (products formed during PBSA biodegradation) during
28 d of degradation with activated sludge. Ando et al. [1]
concluded that any oligomers containing hydroxyl terminals,
such as BSB or BHB, were not detected at all. They speculated
that this was because those oligomers were hydrolyzed quickly
into other products by the action of lipases. However, we have
detected these oligomers in the products. This confirms that
our preparation technique was very useful for detecting a small
amount of minor products. Comparison of the rates of bio-
degradation showed that the diurethane structure of BHB
seemed very stable. The half-life for BSB was estimated as 3
d, but that for BHB was expected to be more than 50 d if the
initial rate of biodegradation was maintained. Owen et al. [4]
reported that the diurethane compound toluene-2,4-dicarbamic
acid, diethyl ester was biodegraded to tolulene-2,4-diamine via
the aromatic amine intermediates carbamic acid, (3-amino-4-
methylphenyl)-, ethyl ester, and carbamic acid, (5-imino-2-
methylphenyl)-, ethyl ester [4]. Analysis of the results of that
study showed that the compounds of urethane groups were
likely modified to the metabolic intermediates and accumu-
lated in the culture before achievement of complete biodeg-
radation. Therefore, we determined a total carbon balance to
confirm the carbon evolved as carbon dioxide [5], the carbon
produced as new biomass, and the carbon transformed into
water-soluble organic metabolites for both BSB and BHB. The
results are shown in Table 2. The biochemically oxidized
amount of carbon (CBOD; mg/L) in the test material introduced
into the test system (carbon content [CMAT; mg/L]) was cal-
culated from the percentage biodegradation. The increase in
biomass carbon in the test reactors containing test material
(CBIO; mg/L) was obtained from carbon analysis of the filter
cakes. The increase in DOC during the incubation period
(CDOC; mg/L) was determined by DOC analysis. The amount
of organic carbon in the residual test substance (CRES; mg/L)
Fig. 1. Composition of the water-soluble products after 5 h and 20 h
of enzymatic degradation of poly(tetramethylene succinate-co-tetra-
methylene adipate) (PBSA) powder and after 20 h of degradation of
poly(tetramethylenesuccinate) (PBS) powder with Chromobacterium
extracellular lipase at 35ЊC and pH 7.5. Each product is represented
by the molecular weight corresponding to the structure in Table 1.
resented as adipate at a terminal, for convenience. Figure 1
shows the composition of water-soluble products during the
enzymatic degradation of PBSA powder. The products range
from 90 to 834 in molecular weight, corresponding to S and
ABSBSBSBS, respectively. The main products after 5 h of the
enzymatic degradation were oligomers of SBSBSB (mol wt
534), SBSBS (mol wt 462), ABSB (mol wt 390), and SBSB
(mol wt 362). On the other hand, the main products after 20
h of the enzymatic degradation were oligomers of ABSB,
SBSB, SBS (mol wt 290), and SB (mol wt 190). Ando et al.
[1] showed that oligomers of one or two ester linkages were
released in nearly equal ratios in the presence of three different
extracellular lipases and were stable. The suggestion has been
made that oligomers of one or two ester linkages would have
low binding affinities for the lipases. The fraction of large
oligomers containing more than three ester linkages in the
products significantly diminished from 5 to 20 h during en-
zymatic degradation. Therefore, oligomers of one, two, or three
ester linkages could be considered as the dominant products.
Table 1 summarizes all the products from PBSA. In these
products, SBHBSBSB (mol wt 792), SBHBSB (mol wt 620),
ABHBS (mol wt 576), SBHBS (mol wt 548), ABHB (mol wt
476), SBHB (mol wt 448), and BHB (mol wt 348) differ struc-
turally from the other polyester-based oligomers with respect
to the diurethane segment. The diurethane segment comes from
hexamethylene diisocyanate (H), which functions as a coupling
agent to increase molecular weight [3]. The resulting PBSA
behaves as a biodegradable plastic. These diurethane products
also underwent enzymatic degradation and finally degraded to
SBHB or BHB after 20 h. Consequently, we obtained 28 spe-
cies of water-soluble products from PBSA and the small olig-
omers such as SB, AB, BSB, SBS, ABS, BHB, SBSB, ABSB,
and SBHB were the major products at the end of enzyme
degradation. We also degraded PBS in the same way and ob-
was determined by SEC-MS analysis. In the case of BSB, CBOD
/
CMAT, CBIO/CMAT, CDOC/CMAT, and CRES/CMAT were approxi-
mately 0.80, 0.05, 0.02, and 0.00, respectively. In the case of
BHB, those same relationships were approximately 0.10, 0.00,
0.90, and 0.20, respectively. This observation suggested that
70 mol% of BHB would be biochemically modified and ac-
cumulated in the culture medium as the water-soluble inter-
mediate metabolites.
Synergic effect in biodegradation
All of the water-soluble products from PBSA present after
20 h of enzymatic degradation were supplied into the activated
sludge at the same time and biodegraded. The percentage bio-
degradation value of each product was calculated as the ratio
of the specific BOD to the theoretical oxygen demand. The
percentage residue products were determined by HPLC-MS
analyses. The total percentage biodegradation value was con-
tinuously recorded and the percentages of residual products
were measured after 1, 2, 3, and 16 d of cultivation and
summed up as shown in Figure 3. Analysis of the results
indicated that all the products were readily biodegradable and
completely extinguished from the culture when the total bio-
degradability reached a constant value (80%). The apparent
half-life for the aggregate is estimated to be 3 d, the same as

944
Environ. Toxicol. Chem. 20, 2001
E. Kitakuni et al.
Table 1. Molecular weight (mol wt) and structure of all the water-soluble products from poly(tetramethylene succinate-co-tetramethylene adipate)
a S
ϭ succinic acid; B ϭ 1,4-butanediol; A ϭ adipic acid; BHB ϭ bis(hydroxybutyl) hexamethylene dicarbamate.

Biodegradation of PBS/PBSA through water-soluble products
Environ. Toxicol. Chem. 20, 2001
945
Fig. 3. Material balance in the biodegradation of all the water-soluble
products prepared from poly(tetramethylene succinate-co-tetramethy-
lene adipate). The percentage biodegradation was calculated from the
amount of biochemical oxygen demand (BOD) and a biodegradation
curve was obtained (dotted line). The total amount of residual products
was measured by high-performance liquid chromatography–mass
Fig. 2. Biodegradation curves of bis(hydroxybutyl) succinate (BSB)
(⅜), bis(hydroxybutyl) hexamethylene dicarbamate (BHB) (ⅷ), and
aniline as a positive reference (dotted line) in the activated sludge.
spectrometry after 1, 3, 5, and 16 d of cultivation (Ⅵ) and the per-
centage residual fraction curve was obtained (solid line).
for single BSB. This observation indicates that the diurethane
products such as BHB and SBHB were readily degraded in
parallel with the other products. We then conducted the bio-
degradation test of BHB in the presence of BSB. The BSB
was supposed to be able to induce a rapid growth of micro-
organisms in the culture and the increase in population was
thought to possibly facilitate the degradation of BHB. The 24
mg of BHB and 6 mg of BSB (equivalent to mole ratio of
BHB:BSB of 3:1) were supplied into the activated sludge and
biodegraded for 28 d. The results are shown in Figure 4. The
BHB and BSB had a biodegradability of 10% or 80%, re-
spectively, when 30 mg of each substance was applied indi-
vidually in the biodegradation test. If BHB and BSB were
biodegraded independently, either would contribute to the per-
the percentage biodegradation of BHB in the presence of BSB
would be expected to be 15% (31%
Ϫ 16%), which is double
that of BHB by itself (8%). The notion of synergism in bio-
degradation was reinforced by the soil burial test of BHB.
Native soil is an inoculum rich in a variety of microorganisms.
Figure 5 shows that the biodegradation of BHB attained a value
centage biodegradation value by 8% (10%
ϫ 24 mg/30 mg)
and 16% (80% 6 mg/30 mg), respectively. The total per-
ϫ
centage biodegradation value was then expected to be 24%.
However, the test revealed that the real measured value reached
31%. The BSB was assumed to be biodegraded rapidly and
the rate of biodegradation was supposed to be limited. If so,
Table 2. Carbon balance of bis(hydroxybutyl) hexamethylene
dicarbamate (BHB) and bis(hydroxybutyl) succinate (BSB).a Percentage
fraction values divided by CMAT are shown in parentheses
Oligo-
mer
CMAT
(mg/L)
CBOD
(mg/L)
CBIO
(mg/L)
CDOC
(mg/L)
CRES
(mg/L)
BSB
BHB
16.47
(100.0%)
16.56
13.31
(80.8%)
1.66
1.89
(11.5%)
ND
0.42
(2.6%)
11.30
ND
3.31
(100.0%)
(10.0%)
(68.2%)
(20.0%)
a CMAT
ϭ
carbon content in the test material introduced into the test
Fig. 4. Biodegradation curve of the blend of bis(hydroxybutyl) suc-
cinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate
(BHB) at a mole ratio of 1:3 as BSB:BHB (actual curve; solid line).
The predicted biodegradation curve (predicted curve; dotted line) was
calculated from the biodegradation curves of single BHB and that of
single BSB assuming that BSB and BHB are independently biode-
graded.
system; CBOD is the biochemically oxidized amount of carbon; CBIO
is the carbon content in the filter cake regarded as biomass carbon;
CDOC is the increase in dissolved organic carbon during incubation
period; CRES is the carbon content in the residual test material de-
termined by size exclusion chromatography and mass spectroscopy;
ND
ϭ not determined.

946
Environ. Toxicol. Chem. 20, 2001
E. Kitakuni et al.
Conclusion
The water-soluble products from enzymatic hydrolysis of
PBSA or PBS are classified into polyester-based products and
diurethane products. The formers have half-lives of about 3
d. On the other hand, the latter are biodegraded at a much
slower rate. However, the polyester-based products likely en-
hance the activities of microorganisms in a degradation en-
vironment and thereby facilitate the biodegradation of the di-
urethane products. We showed that a few small products were
preferentially digested, meaning that microorganisms could
select them in proportion to the molecular sizes and utilize the
polyester in preference to the polyurethane. These products
would be metabolized in the cell and become oxidized. With
regards to their metabolites, the small products such as BSB
and BHB were important as the starting material in the met-
abolic pathway. The BHB seemed to undergo ␤-oxidation and
be transformed. The significant feature of this work is its in-
dication of the environmental friendliness of PBSA and PBS.
This approach could be applied to other biodegradable poly-
mers.
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is about 15 d, which is comparable to that of poly(3-hydrox-
ybutylate) as a positive reference. The difference in half-lives
between BHB (30 d) and PBSA (15 d) suggests that the readily
biodegradable substances such as BSB have an important role
in promoting PBSA biodegradation.